The Enhancer of split complex

Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors that are components of the Notch signaling
pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Using random oligonucleotide selection, the optimal DNA binding site for the Enhancer of split proteins has been determined to be a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains
an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate
similar target genes in vivo. The 3 base pairs flanking the E-box core are intrinsic to DNA recognition by these proteins and the Enhancer of split
and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three
closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of
proteins that can bind in vivo (Jennings, 1999).

The three related sequences studied were the B1, A1 and A2 sites. The B1 and A1 sites contain optimal flanking bases and differ by a single-base substitution that switches the E box from a class B site (B1) to a class A site (A1). The A2 site has the class A E-box core but suboptimal flanking sequences. The consensus binding site for the E(spl)bHLH proteins contains a
class B canonical E-box (CACGTG). This is compatible with
the presence of the key arginine residue in the basic region that is
characteristic of all other bHLH proteins that recognize class B sites and which contacts the central G. The
selected site differs from the previously identified N box (CANNAG),
indicating that the latter may not be generally representative of
E(spl) target sites -- in vitro, the N box is a much
lower affinity target that the class B site. The 12-bp palindrome
(the optimal site) is termed the ESE box. Flanking bases have
been implicated in DNA binding by other bHLH proteins, including c-Myc and Hairy, based on in vitro assays and X-ray crystallography studies that reveal interactions between bHLH
proteins and bases outside the E-box core. However, the flanking bases preferred by c-Myc and Hairy differ from
those selected by E(spl)bHLH proteins, indicating that in vivo, the sequences immediately surrounding an E box
are important for determining exactly which bHLH proteins bind there to
regulate transcription (Jennings, 1999 and references).

The interactions with flanking bases helps to explain the specificity
in vivo of different bHLH proteins, an important factor given the large
number of bHLH proteins identified to date. The in vivo expression
patterns produced by E boxes with different flanking bases in these
experiments emphasizes the significance of the flanking sequences. For example, a comparison
between the A2 and A1 sites demonstrates that the former is a target
for many more transcriptional activators. These experiments also
illustrate the relevance of different E-box core sequences, since a
single-base difference within the E-box core (A1 to B1) is sufficient
to prevent binding of proneural proteins and other activators. This is
in agreement with earlier studies that argued that
proneural proteins and E(spl)bHLH repressors recognize sites with
distinct types of E-box cores. However, these results show that
E(spl)bHLH repressors prefer the class B core, which is recognized
by many different bHLH activators and repressors, over the class C
core. Class C has been designated the target for repressor bHLH proteins that
contain a proline residue in the basic domain. The
class C site (CACGCG) is the optimal binding site for the
Drosophila Hairy protein, whose basic
domain contains a proline residue but differs from E(spl)bHLH
proteins in 7 of the 11 remaining residues, which could account for the
different profile of DNA binding specificities. The distinctions in the
DNA binding specificities could be significant for studies of the
vertebrate homologs of the E(spl)bHLHs and Hairy. Overall, the in
vitro binding experiments and the activity of different sites in vivo
demonstrate that the bHLH proteins that were tested can recognize a
specific range of target sequences and that both core and flanking
bases are important for determining the binding specificity (Jennings, 1999 and references).

Although flanking bases may distinguish sites for
different types of E-box binding proteins, there are no significant
differences in the bases recognized by individual E(spl) proteins; the
same consensus binding site was derived for each of three proteins tested. There were subtle differences in the ranges of
oligonucleotides, with Mdelta selecting a broader range of variants at
the flanking sites than Mgamma and M3 and the latter two proteins
exhibiting more tolerance for variants in the core E box, but
experiments comparing the affinity of the proteins for these variant
sites reveal no detectable bias (Jennings, 1999).

The binding specificities observed are all for homodimers of individual
E(spl) proteins. In places where more than one E(spl)bHLH protein
is expressed (e.g., proneural clusters), it is possible that the
proteins form heterodimers among themselves to bind DNA and repress
transcription. However, given that the amino acid sequences of the DNA
binding domains and the DNA binding preferences of the individual
E(spl)bHLH proteins are so similar, it seems unlikely that
heterodimers between E(spl)bHLH proteins would differ greatly from homodimers in their DNA binding sequence
preferences. In addition, during several developmental processes, a
single E(spl)bHLH protein predominates (e.g., Mbeta in the
presumptive intervein region of the wing),
indicating that E(spl)bHLH proteins are likely to function as do homodimers. There is
also no evidence to suggest that the E(spl)bHLH proteins are
required to form heterodimers with other bHLH family members to bind
DNA and repress gene transcription in response to Notch signaling.
Thus, the homodimers analyzed in these experiments likely represent
complexes that are functional in vivo (Jennings, 1999 and references).

The overall similarity in the binding of different E(spl) proteins in
vitro suggests that they are capable of recognizing the same targets in
vivo and is consistent with the phenotypes observed when the individual
proteins are expressed ectopically. Ectopic expression of M8, M5, Mbeta,
Mdelta, and M7 all produce phenotypes of vein and bristle loss. Both Mbeta and M7 are
able to interact with DNA sequences regulating achaete. The ability to recognize the same DNA target sequences
could explain the apparent redundancy between the E(spl)
genes, as they would all have the potential to act
in the same processes. The observation that specific E(spl)bHLH
proteins are more or less efficient in regulating different processes (e.g., Mbeta more effective at suppressing veins and M8 more effective at
suppressing bristles) is thus more likely to be
consequence of differences in protein:protein interactions than of
differences in target recognition (Jennings, 1999 and references).

In the absence of E(spl)bHLH proteins, proneural
protein expression persists at high levels in all cells of a proneural
cluster. Thus, one action of E(spl)bHLH proteins is
to antagonize the proneural proteins, with the ultimate consequence
that proneural gene expression is repressed. It has been proposed that
E(spl)bHLH proteins exert their influence by binding to regulatory
regions within the AS-C and repressing transcription of the
proneural genes. This hypothesis is
supported by the observations that expression of Achaete is induced by
M7ACT and MbetaACT and
that induction of ectopic bristles in the Drosophila wing
and notum by M7ACT is abolished in the absence of proneural
proteins. One putative binding site for the
E(spl)bHLH proteins, that upstream of the achaete gene, has
the sequence 5'-CGGCACGCGACA-3' (Hairy site).
Mgamma will bind this site in vitro, and M7 can bind this
sequence and repress transcription in a cotransfection assay in
Drosophila S2 cultured cells. However,
mutation of this site in vivo results in a phenotype resembling that
caused by mutations in hairy rather than in the
E(spl)-C. This fits with the observation
that this sequence conforms to an optimal Hairy DNA binding site but is
a suboptimal site for the E(spl) proteins and indicates that
the E(spl) proteins do not recognize this sequence in vivo. Thus, if
E(spl) proteins are directly repressing achaete expression,
there should be more optimal target sites elsewhere within the
AS-C. Indeed, a search of recently available AS-C
genomic sequence identifies >10 sequences with good
matches to ESE boxes, in
addition to the sites that have been identified by in vitro binding
assays (Jennings, 1999 and references).

An alternative hypothesis is that the primary function of the
E(spl)bHLH proteins is to antagonize the actions of proneural proteins posttranscriptionally. Evidence in support of this comes from
experiments in which L'sc is ectopically expressed using a
heterologous promoter that is not subject to direct regulation by
E(spl)bHLH proteins. Under these conditions L'sc
expression results in isolated ectopic bristles, rather than clusters
of bristles, demonstrating that lateral inhibition is still able to
restrict neural fate to a single cell even though l'sc
transcription is insensitive to Notch signaling. This implies that
E(spl)bHLH proteins are able to antagonize proneural genes in ways other
than by repressing their transcription. One possibility is that the E(spl) proteins can interact with the same targets as proneural proteins, but that they repress rather than activate transcription. The
ability of E(spl) proteins to bind to the B1 and A1 sequences and
repress transcription from a heterologous promoter is consistent with
this model, as is the observation that M7ACT can induce
certain ectopic leg bristles in the absence of the achaete
and scute genes. In the latter context,
M7ACT is likely to be acting on genes with functions
downstream of the proneural proteins to cause neural differentiation.
In addition, the E(spl)bHLH proteins are involved with
developmental processes that do not involve the proneural proteins,
e.g. wing vein development; thus, they cannot act solely to repress
proneural gene transcription during development (Jennings, 1999 and references).

How might E(spl)bHLH repress transcription of target genes? The
closely related protein Hairy has been shown to repress transcription in a dominant manner even when its binding sites are located at some
distance from the promoter, leading to the hypothesis that Hairy is able to mediate stable, inheritable repression of the
target genes. It is anticipated that E(spl)bHLH repression will be
transitory, so that if Notch signaling were terminated, the E(spl)
proteins would decay and the target genes would be susceptible to
reactivation. Although proneural and E(spl)bHLH proteins optimally prefer different core E-box binding sites, so that independent binding
to target genes appears likely, the importance of the bases flanking
the E box in target recognition means that there is potential for
overlap in the binding sites of the two groups of proteins. Thus, in
cells where expression of E(spl)bHLH proteins is induced by Notch
signaling, the proteins accumulate to high levels and could compete
for binding to proneural protein target sites of the A1 type described
here. Among the E-box sequences recognized by proneural proteins in
vitro that have been described, at least a subset have good matches
with the ESE consensus and thus could be recognized by both classes of
proteins. Now that
the sequence preferences of the E(spl)bHLH proteins have been identified, when target
genes of proneural and E(spl)bHLH proteins have also been identified and
their regulatory regions analyzed, it will be possible to determine
whether the sites present offer the potential for competition (e.g., by
resembling A1 sites) or whether they have the features of
completely distinct binding sites for E(spl)bHLH, Hairy, proneural,
and other bHLH proteins (Jennings, 1999 and references).

It is proposed that Hairless prevents ISC loss by repressing expression of Notch target genes, including the E(spl)-C genes. It is further proposed that Da-dependent bHLH activity promotes ISC identity, including the ability to self-renew and to express Delta. Delta, in turn, activates Notch in the adjacent EB, releasing the intracellular domain of Notch (NICD). It is speculated that, in response to Notch activation, the E(spl)-bHLH repressors downregulate Da-dependent bHLH activity in EBs as described in other systems, thereby shutting off ISC identity and promoting differentiation (Bardin, 2010).

E(spl)-C bHLH repressors act in part through their ability to inhibit bHLH activators. The data demonstrate that Da is also essential to maintain ISC fate and that E-box Da-binding sites are required to promote ISC-specific enhancer activity. Thus, it is proposed that activation of E(spl)-C genes by Notch in EBs downregulates Da bHLH activity and thereby contributes to turning off ISC identity in the differentiating cell (see Model for ISC maintenance). The specificity of ISC-specific E-box expression might be due to the ISC-specific expression of a bHLH family member. Although an array analysis raised the possibility that Scute may be specifically expressed in ISCs, genetic analysis indicates that scute function is not essential for ISC maintenance. Alternatively, specificity of gene expression might result from inhibition of bHLH activity in the EB and differentiating daughters, possibly by E(spl)-bHLH factors, rather than by the ISC-specific expression of a Da partner. It is also possible that a non-bHLH, ISC-specific factor restricts the Da-dependent bHLH activity to ISCs in a manner similar to the synergism observed in wing margin sensory organ precursors (SOPs) between the Zn-finger transcription factor Senseless and Da (Bardin, 2010).

Recently, a role for the Da homologs E2A (Tcf3) and HEB (Tcf12) has been found in mammalian ISCs marked by the expression of Lgr5 and, in this context, E2A and HEB are thought to heterodimerize with achaete-scute like 2 (Ascl2), which is essential for the maintenance and/or identity of Lgr5+ ISCs (van der Flier, 2009). In Drosophila, however, AS-C genes are not essential for ISC maintenance, but appear to play a role in enteroendocrine fate specification. The observation that Da bHLH activity is required for the identity of both Drosophila ISCs and mammalian Lgr5+ ISCs suggests that there might be conservation at the level of the gene expression program. Additionally, the bHLH genes Atoh1 (Math1) and Neurog3 are both important for differentiation of secretory cells in the mammalian intestine. Clearly, further analysis of the control of Da/E2A bHLH activity, as well as of the gene networks downstream of Da/E2A, will be of great interest (Bardin, 2010).

The data suggest that ISC fate is promoted both by inhibition of Notch target genes through Hairless/Su(H) repression and by activation of ISC-specific genes through bHLH activity. How then is asymmetry in Notch activity eventually established between the two ISC daughters to allow one cell to remain an ISC and one cell to differentiate? Three types of mechanisms can be envisioned that would allow for asymmetry of Notch signaling (Bardin, 2010).

First, the binary decision between the ISC and EB fates might result from a competition process akin to lateral inhibition for the selection of SOPs. In this process, feedback loops establish directionality by amplifying stochastic fluctuations in signaling between equivalent cells into a robust unidirectional signal. The finding that the Da activator and E(spl)-bHLH repressors are important to properly resolve ISC/EB fate is consistent with this type of model. Activation of the Notch pathway in one of the daughter cells may then lead to the changes in nuclear position (Bardin, 2010).

Second, the asymmetric segregation of determinants could bias Notch-mediated cell fate decisions. The cell fate determinants Numb and Neur are asymmetrically segregated in neural progenitor cells to control Notch signaling. However, no evidence was found for the asymmetric segregation of these proteins in dividing ISCs. Additionally, the data indicate that Numb is not important to maintain ISC fate. It cannot be excluded, however, that another, unknown Notch regulator is asymmetrically segregated to regulate the fate of the two ISC daughters (Bardin, 2010).

A third possibility is that after ISC division, one of the two daughter cells receives a signal that promotes differential regulation of Notch. Indeed, it has been noted that the axis of ISC division is tilted relative to the basement membrane, resulting in one of the progeny maintaining greater basal contact than the other. An extracellular signal coming either basally or apically could bias the Notch-mediated ISC versus EB fate decision. For instance, Wg secreted by muscle cells could act as a basal signal to counteract Notch receptor signaling activity in presumptive ISCs. This could be accomplished by Wg promoting bHLH activity or gene expression. Indeed, Wg has been demonstrated to promote proneural bHLH activity in Drosophila (Bardin, 2010 and references therein).

These models are not mutually exclusive, however, and proper control of ISC and differentiated cell fates during tissue homeostasis might involve multiple mechanisms (Bardin, 2010).